Project Summary More than 30 autoimmune type 1 diabetes (T1D) susceptibility loci (termed Idd) have been identified in the nonobese diabetic (NOD) mouse, a spontaneous animal model for the human disease. Among those, the Idd3 locus has been mapped to a 650 kb region on Chromosome 3 containing five protein-coding and two predicted genes, of which Il-2 and Il-21 are top candidates. The C57BL/6 (B6)-derived Idd3 confers strong T1D resistance, whereas the NOD-derived interval contributes to disease development. Remarkably, the IL-2/IL-21 locus has also been mapped for human T1D in genome-wide association studies. Due to the tight linkage between Il-2 and Il-21 genes (100kb apart), congenic strains that respectively carry only B6-derived Il-2 or Il-21 gene have not been made available to independently test if either or both of them represent the Idd3 underlying genes. Initial studies that involved haplotype mapping and expression analyses provided evidence to support Il-2 as the Idd3 gene. Recently, a diabetogenic role of interleukin (IL)-21 has also emerged, and a higher level of this cytokine was shown to be expressed by NOD mice compared to the NOD.Idd3B6 congenic strain. Since allelic variations in both Il-2 and Il-21 genes exist between NOD and B6 strains, a possibility tha both genes contribute to the T1D regulatory function of the Idd3 locus cannot be excluded. However, previous studies lacked the appropriate genetic tools to independently and definitively test if Il-2 and/or Il-21 are the casual genes within the Idd3 region. In this application, we propose to use zinc-finger nucleases (ZFNs) to establish a mouse model system where a pair of F1 hybrid strains respectively express only one of the two parental alleles of a candidate gene, but are genetically identical throughout the genome to directly determine the role of the implicated disease gene. The ability of the ZFN technology to specifically knock out a gene in mouse strains lacking germ-line transmittable embryonic stem cells will allow us to target both Il-2 and Il-21 in standard NOD mice and the NOD.Idd3B6 congenic strain. Our goal is to determine if Il-2 or Il-21, or both are the Idd3 underlying genes by establishing a series of (NOD x NOD.Idd3B6) F1 strains where only one parental allele of Il-2 or Il-21 (NOD or B6) is expressed.